Genetically shaping morphology of the filamentous fungus Aspergillus glaucus for production of antitumor polyketide aspergiolide A.
Genetically shaping morphology of the filamentous fungus Aspergillus glaucus for production of antitumor polyketide aspergiolide A.
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用于生产抗肿瘤聚酮曲霉内酯 A 的丝状真菌青曲霉形态的遗传塑造
DOI：
10.1186/1475-2859-13-73
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发表时间：
2014-05-20
影响因子：
6.4
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Cai M;Zhang Y;Hu W;Shen W;Yu Z;Zhou W;Jiang T;Zhou X;Zhang Y
研究背景对于丝状真菌而言，菌丝作为基本生长单位，通常对生物反应器中机械力和流体动力产生的剪切应力非常敏感，严重影响了微生物产量。针对真菌发酵中的剪切敏感难题，传统的解决方法主要是采用搅拌、桨型和反应器结构，这给工业生产带来了成本高、工作坚韧的问题。本研究旨在对剪切敏感型丝状真菌灰绿曲霉进行抗剪切形态的遗传改造，使其适应生物反应器，从而建立高效的发酵工艺。应用glaucus以降低其剪切敏感性并提高曲霉素A的产量。采用简并PCR和基因组步移法获得极性生长基因AgkipA和AgteaR，通过同源整合双交换构建基因缺失突变体。形态学分析表明，ΔAgkipA和Δ AgteaR基因的缺失导致菌丝弯曲，而ΔAgkipA基因的缺失导致菌丝弯曲的程度比ΔAgteaR基因的缺失小。突变体分生孢子萌发第二根芽管变得随机，而菌落生长发育基本保持不变。ΔAgkipA和ΔAgteaR突变体在液体培养中的形态分别为致密的颗粒和疏松的团块。与野生型菌株相比，两种突变体弯曲菌丝对玻璃珠研磨的抗性均不显著。然而，它们产生了非常不同的肉汤流变学，这进一步导致生物反应器发酵中的生长和代谢变化。ΔAgkipA突变株通过形成小球，创造了一个低粘度、低剪切力和高溶解氧张力的罐内环境，使曲霉素A的产量（121.7 ± 2.3 mg/L）比野生型提高了82.2%。本研究为通过基因改造实现深层发酵真菌形态的塑造提供了有用的信息，对工业丝状真菌形态改良具有重要的参考价值。
BackgroundFor filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in bioreactor, and it severely decreases microbial productions. The conventional strategies against shear-sensitive conundrum in fungal fermentation usually focus on adapting agitation, impeller type and bioreactor configuration, which brings high cost and tough work in industry. This study aims to genetically shape shear resistant morphology of shear-sensitive filamentous fungus Aspergillus glaucus to make it adapt to bioreactor so as to establish an efficient fermentation process.ResultsHyphal morphology shaping by modifying polarized growth genes of A. glaucus was applied to reduce its shear-sensitivity and enhance aspergiolide A production. Degenerate PCR and genome walking were used to obtain polarized growth genes AgkipA and AgteaR, followed by construction of gene-deficient mutants by homologous integration of double crossover. Deletion of both genes caused meandering hyphae, for which, ΔAgkipA led to small but intense curves comparing with ΔAgteaR by morphology analysis. The germination of a second germ tube from conidiospore of the mutants became random while colony growth and development almost maintained the same. Morphology of ΔAgkipA and ΔAgteaR mutants turned to be compact pellet and loose clump in liquid culture, respectively. The curved hyphae of both mutants showed no remarkably resistant to glass bead grinding comparing with the wild type strain. However, they generated greatly different broth rheology which further caused growth and metabolism variations in bioreactor fermentations. By forming pellets, the ΔAgkipA mutant created a tank environment with low-viscosity, low shear stress and high dissolved oxygen tension, leading to high production of aspergiolide A (121.7 ± 2.3 mg/L), which was 82.2% higher than the wild type.ConclusionsA new strategy for shaping fungal morphology by modifying polarized growth genes was applied in submerged fermentation in bioreactor. This work provides useful information of shaping fungal morphology for submerged fermentation by genetically modification, which could be valuable for morphology improvement of industrial filamentous fungi.
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通讯作者：
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